Entamoeba histolytica is a protozoan parasite capable of producing serious and even fatal disease in humans. The mechanism(s) whereby E. histolytica trophozoites produce cell and tissue damage and diarrhea are unknown. Since diffuse tissue damage is seen in human amebic colitis, often in the absence of trophozoites, we searched for and detected an extractable cytotoxic substance in axenically reared E. histolytica trophozoites. The toxic properties of the cytotoxin were inhibited by alpha-globulins known to inhibit proteases. Comparison of strains of decreasing virulence revealed greater concentrations of cytotoxin in the more virulent strains. Release of cytotoxin into serum free culture medium was demonstrated and shown to be parallelled by release into culture medium of cathepsin-line protease activity. Specific inhibitors and activators of the thiol-proteinase activity have similar effects on the tytotoxic activity of the partially amebal cytotoxin. The aims of this phase of our research are to utilize purification methods for cathepsins to obtain highly purified preparations of amebal cytotoxin/cathepsin and to use such preparations to better characterize the mechanism(s) by which this substance(s) might play a role in the virulence of the organism. Specific antibody raised to the purified toxins will be used to localize the toxic substances within and at the sufaces of trophozoites from cultures and in tissues from experimental amebic colitis and liver abscess in animal models. The mode of action of the cytotoxin/cathepsin will be studied in a number of assay systems. The enterotoxic activity of E. histolytica trophozoites will be independently purified, and cytotoxin/cathepsin and enterotoxin will be assayed for enterotoxicity using and in vivo perfused jejunal loop method in rabbits. The relationship of the purified enterotoxin to the cytotoxin/cathepsin will be determined. These studies should help to eluciate the basic pathogenic mechanisms by which this protozoan parasite causes disease. A better understanding of pathogenesis should stimulate new, more effective means of prevention, diagnosis and treatment of amebiasis.